Clefts of the lip and palate are among the most common craniofacial birth defects. They can co-occur with other symptoms as part of Mendelian disorders, but the majority of cases are non-syndromic and have a complex etiology. In some cases disrupted protein-coding genes have been identified as contributors to orofacial clefting risk. However, accumulating evidence from genome-wide association studies (GWAS) indicates that sequence variation in non-coding regions also strongly contributes to a variety of clinical disorders including orofacial clefting. While it is speculated that many of these variants affect disease through impacting on functional properties of distant-acting transcriptional enhancers, only very few isolated examples of such regulatory variation have been identified. This is likely due to the fact that the genomic location and function of the vast majority of distant-acting enhancers in the human genome remains unknown. To address the pressing need to identify on a genomic scale enhancers that are involved in face and palate development and likely relevant for clefting etiology, we propose here an integrated genomic and transgenic mouse strategy to identify craniofacial enhancers and characterize their activities. Specifically, we will use a ChlP-seq approach to identify genome-wide sets of enhancers that are active in mouse face and palate tissues at embryonic stages that are relevant for orofacial clefting. We will use a transgenic mouse enhancer screen to validate and characterize 130 of these enhancer predictions in detail by determining their in vivo activity patterns. Furthermore, we will identify disease-associated GWAS variants that map to craniofacial enhancers that we will have discovered. We will then test and compare the variant and normal sequences in the transgenic enhancer assay for differences in their in vivo activities. All of the genomic and in vivo datasets, as well as molecular reagents developed through these experiments will be made available to other investigators through the Face Base program in order to maximize their availability and accelerate the progress of biomedical and clinical studies of mid-face and palate development and orofacial clefting.